DE69529665T2 - HORMONE ANALOGS CONTAINING BOR AND METHOD FOR THEIR USE FOR IMAGING OR KILLING CELLS THAT HAVE HORMONE RECEPTORS - Google Patents

Abstract

Boron containing compounds are targeted to hormonally responsive cells by contacting the cells with a conjugate of a boron containing moiety conjugated to a ligand having binding specificity for an intracellular receptor of the cell. The conjugates are useful for imaging or killing hormonally responsive cells, such as hormonally responsive tumor cells. For target cell killing, the cells are contacted with a 10B containing moiety conjugated to a ligand having binding specificity for an intracellular hormone receptor of the cells. The 10B containing moiety becomes associated with the hormone receptor of the cells, which may then be irradiated with neutrons to kill the cells by boron neutron capture. For target cell imaging, the cells are contacted with a 11B containing moiety conjugated to a ligand having binding specificity for an intracellular hormone receptor of the cells, and the boron that becomes associated with the hormone receptor of the cells may then be imaged using magnetic resonance imaging techniques.

Description

The present invention relates to new agents for use in boron imaging and boron neutron capture therapy. In particular, this invention relates to boron neutron capture or imaging agents that involve a unit containing boron is conjugated to a ligand, the binding specificity for one Has a cell hormone receptor.

background the invention

The development of quantum mechanics, nuclear physics and the associated chemistry that began in the 1930s led James Chadwick to discover the neutron in 1932 (Nature 129, 312, 1932). Studies of the interactions of neutrons with a large number of materials led to the discovery of the phenomenon of neutron scattering by elastic collisions (JR Dunning et al., Phys. Rev. 47, 325, 1935) with atomic nuclei, in particular with the proton of the H atom. The capture of slow (or thermal) neutrons by certain nuclei has been disclosed in Fermi et al., Proc. Roy. Soc. London 146, 483 (1934); and the decay of other special nuclei by interaction with thermal neutrons was disclosed in JR Dunning et al., Phys. Rev. 48, 265 (1935). A huge amount of experimental data had already been collected by 1935, showing that the ability of an atomic nucleus to capture a neutron was not related to the mass of the target nucleus, but to the actual structure of that nucleus. The idea of cores with a characteristic cross-sectional area, expressed as units of 10 ^-24 cm ² , which are known as Barn units, was introduced in the course of this early work. It was known that the core cross section of boron for neutron capture was exceptionally large, while the neighbors of boron in the periodic table, nitrogen and carbon, had core cross sections that were quite small in comparison.

Taylor, Proc. Roy. Soc. A47, 873 (1935) described the capture of thermal neutrons by ¹⁰ B nuclei, followed by the formation of ⁴ He ²⁺ (? -Particles) and ⁷ Li ³⁺ with a kinetic energy of about 2 MeV on these two Heavy ion products is distributed. It was also found (Taylor, see above) that the translation range of the product ions was particularly short: about 7.6 μm in photographic gelatin and 1.1 cm in air. Consequently, the products lithium-ion and α-particles are short-lived, energetic species that can cause considerable local damage to organic materials through ionization processes.

Gordon L. Locher of the Bartol Research Foundation at the Franklin Institute in Philadelphia, Pennsylvania, recognized the potential medical applications for neutrons and boron neutron capture, Am. J. Roentgenol. and Radium Therapy, 36, 1 (1936). Locher's concept is based on a simple boron neutron capture reaction as the basis of a binary therapeutic procedure in which a ¹⁰ B nucleus, which is contained in a compound that is specifically found in tumors, reacts with a thermal neutron to form the energetic cytotoxic reaction products α- Particles and lithium ion. No radioactive substances are involved in this process, and the therapeutic process can be modulated by controlling the neutron supply in place of the tumor.

The two components necessary for the boron neutron capture (BNC) process, a controllable source of low-energy neutrons with a usable high flux, and suitable boron compounds for tumor localization were still unknown in 1936, and Locher's idea remained prophetic until nuclear reactors became available , which were used to carry out the first experimental tests with thermal neutrons. This initial event did not come until 1954 when Sweet, Farr and their co-workers (M. Javid et al., J. Clin. Invest. 31, 603 (1952); WH Sweet, N. Engl. J. Med. 245, 875 (1951 ); WH Sweet and M. Javid, J. Neurosurg. 9, 200 (1952); LE Farr et al., Am. J. Roentgenol. 71, 279 (1954); JT Godwin et al., Cancer 8, 601 ( 1955)) treated human brain tumors (glioblastoma multiforme) in end-stage patients with ¹⁰ B-enriched borate as the ¹⁰ B target species. In these first experiments, BNCT was applied to the problem of killing malignant glioma cells that remained at the tumor site after the usual surgical procedures.

The reaction of boron neutron capture (BNC) obtained with thermal neutrons at 293 K (0.025 eV) can be represented as shown in equation (1):

The ¹¹ B core cannot undergo a BNC reaction, while the core cross section of ¹⁰ B is 3837 Barn.

Two other nuclides, ¹ H and ¹⁴ N, are common in tissue, participate in the key neutron capture side reactions that occur with BNCT, and thus contribute significant doses of background radiation to the patient. These two neutron capture reactions do not play a role because of a higher core cross-section of the target nuclei, but because of their very high concentrations in the tissue. As disclosed by H. Hatanaka, Boron-Neufron Capture Therapy for Tumors (H. Hatanaka, ed.), Nichamura Co. Ltd., Nigata, Japan, p. 5 (1986), the neutron capture reactions of ¹ N and ¹⁴ N are as shown in equations (2a) and (2b):

The passage of a neutron through hydrogen-rich media, such as tissue, leads to the deceleration and scattering of these neutrons by collisions with nuclear protons of the H atom. Occasionally, a slow-moving neutron is captured by such a proton and creates a deuteron, accompanied by a characteristic gamma radiation that contributes to the total radiation dose. In another competing capture reaction, the nitrogen atoms present in the tissue can capture a low-energy neutron and generate ¹⁴ C and a proton with 0.63 MeV (kinetic energy). The kinetic energy which is given to the ions ⁷ Li ³⁺ and ⁴ He ²⁺ resulting from the BNC reaction, as well as that which is connected in a similar manner to the proton and the? Photons, as in (2a) and (2b) are shown, is transferred to the surrounding media. Since all of these energetic nuclear reaction products, with the exception of the? -Photoneu, are heavy particles, the kinetic energy is transferred quickly and takes a very short distance. The rate of linear energy transfer, LET, of these particles is characteristically high and the enormous energy of these reactions is therefore released in a very small volume. For example, the ions formed in the BNC reaction produce ⁷ Li ³⁺ and ⁴ He ²⁺ ionization traces of approximately 0.01 mm in length or corresponding to approximately one cell diameter. Thus, the high LETs that are characteristic of particles that arise from the nuclear reactions taking place in the tissue are particularly lethal for the affected cells due to the high density of the energy given off.

Ideally, those cells that carry a large number of ¹⁰ B nuclei are destroyed by the BNC, while neighboring cells that are ¹⁰ B free are, apart from the contribution of the background reactions ¹ H (n, γ) ² H and ¹⁴ N (n, p) ¹⁴ C are spared. In order for the introduction of ¹⁰ B into the tumor cells to achieve this desired effect in BNCT, the selectivity of the boron introduction into tumor tissue compared to normal tissue which is irradiated with neutrons should be as high as possible. In addition, the actual concentration of ¹⁰ B in the tumor must be high enough to give a local binary therapeutic effect that is significantly above the background radiation dose caused by the neutron capture processes shown above ¹ H (n, γ) ² H and ¹⁴ N (n , p) 14C is delivered. Depending on the exact position of the ¹⁰ B with regard to vital components of the tumor cell structure, the generally recognized minimum ¹⁰ B concentration required for an effective BNC was usually assumed to be between 10 and 30 μg ¹⁰ B / g tumor. If the position of the ¹⁰ B nuclei changes from the outer cell wall to the cytoplasm and to the nucleus of the cell, the ¹⁰ B concentration required for an effective BNCT decreases as expected. For example, ¹⁰ B or higher concentrations may be required for cell wall-bound ¹⁰ B, while only 10 ppm or less is required for ¹⁰ B, which is located in the nucleus of the tumor cell. Another factor is the equilibrium concentration of the thermal neutrons in the target volume of the tissue, since very low neutron intensities require proportionally longer irradiation times to give the required number of BNC events for effective therapy.

Since it is necessary to reach basal levels of tumor cell-associated boron, attempts have been made earlier to increase the supply of boron in tumor cells in order to obtain sufficiently high boron concentrations in the cells for effective boron neutron capture therapy, while using relatively small amounts remain on background boron, which can lead to damage to surrounding cells or tissues when exposed to neutrons. Some strategies for targeting tumor cells use, for example, boron compounds with a certain natural affinity for tumors, such as 4- (dihydroxyboryl) phenylalanine (BPA) or the mercaptoundecahydroclosododecaborate dianion (B ₁₂ H ₁₁ SH ² ; BSH), or species containing boron are attached to other molecules such as e.g. B. Porphyrins attached. In addition, it has previously been proposed to conjugate ¹⁰ B-enriched boron to antibodies used for tumorasso cited antigens are specific in order to improve the therapeutic efficacy of BNC therapy. See, for example, D. Goldenberg et al., "Neutron-capture Therapy of Human Cancer: In vivo Results on Tu mor Localization of Boron-10-Labeled Antibodies to Carcinoembryonic Antigen in the GW-39 Tumor Model System", Proc. Natl. Acad. Sci. 81: 560-563 (1984); R. Barth et al., "Conjugation, Purification and Characterization of Boronated Monoclonal Antibodies For Use in Neutron Capture Therapy", Strahlenther. Oncol. 165 (2/3), 142-145 (1989); S. Tamat et al., "Boronated Monoclonal Antibodies for Potential Neutron Capture Therapy of Malignant Melanoma and Leukemia", Strahlenther. Oncol. 165 (2/3), 145-147 (1989); R. Abraham et al., "Boronated Antibodies for Neutron Capture Therapy", Strahlenther. Oncol. 165 (2/3), 148-151 (1989); A. Varadarajan et al., "Novel Carboranyl Amino Acids and Peptides: Reagents for Antibody Modification and Subsequent Neutron Capture Studies", Bioconjugate Chem. 2 (4), 242-253 (1991); and R. Paxton et al., "Carboranyl Peptide-Antibody Conjugates for Neutron-Capture Therapy: Preparation, Characterization, and In Vivo Evaluation", Bioconjugate Chem. 3 (3), 241-247 (1991). As an alternative, it has been proposed to introduce boron agents into liposome vesicles in order to provide the agents with a longer circulatory life, to protect the agents against attacks by normal physiological agents and to reduce possible toxic side effects. Certain liposomes also specifically target neoplastic tissues. See, for example, "Model Studies Directed Toward The Boron Neutron-Capture Therapy of Cancer: Boron Delivery To Murine Tumors With Liposomes", Proc. Natl. Acad. Sci., USA, 89, 9039-9043 (1993).

A work published in The Prostate 5, 159-180 (1984) entitled "Susceptibility of the Prostate Cancer Cell to Different Physical, Hormonal and Chemical Agents: Present Status and Theoretical Prospects for Improved Prostate Cancer Therapy", written by Oscar Hechter , discusses the possibilities and limitations of using steroid ligands to introduce boron units into specific cells for subsequent boron neutron capture therapy. This work suggests the use of a single carborane cage that contains nine or ten ¹⁰ B atoms per cage. Also discussed is the difficulty in obtaining chemical specificity when using steroid ligands due to the presence of steroid receptors on normal tissue.

In one in Strahlenther. Oncol. 165: 125-126 (1989) (No. 2/3) in the name of Wongwiechintana et al. published Work entitled "Neutron Capture Therapy of Cervical Carcinoma" is the synthesis of biomolecules discussed, the for neutron capture therapy is suitable, and in particular will discloses a hormone compound containing boron containing a boron atom.

A publication by Thomas W. Griffin in Critical Reviews in Oncology / Hematology 1992, 13, 17–31, with the title "Fast Neutron Radiation Therapy" gives an overview on the background of boron neutron capture therapy. In this overview will meet the requirements for discussed successful boron neutron capture therapy, and it will also be possible carrier for one more specific introduction suggested by boron in certain cells.

The international patent publication No. WO 95/26359 on behalf of Regents from the University of California discloses phosphate-based boron-rich oligomers that are based on aminoprotein Delivery systems or regulatory peptides are conjugated.

In a publication by Jorgen Carlsson et al. in International Journal of Radiation Oncology, Volume 30, No. 1, pp. 105-115 (1994), entitled "Strategy for Boron Neutron Capture Therapy against tumor cells with over-expression of the epidermal growth Factor Receptor ", is the conjugation of boron units with growth factor / dextran conjugates discussed, then in boron neutron capture therapy can be used and the improved cellular Retention of the compound and therefore longer exposure times of the subsequently applied Therapy result.

The one transferred to the Indiana University Foundation U.S. Patent No. 4,466,952 discloses a single carborane cage in U.S. Pat 17? Position of estradiol and its subsequent use in boron neutron capture therapy.

Despite the progress made in the Method of introducing boron problems with low cycle life prevent relatively low tumor specificity, not optimal liposome composition, potentially more toxic Side effects and / or suboptimal cell membrane interactions, that these Methods the highly specific introduction of boron into target tumor cells in therapeutically desirable Achieve concentrations. There is therefore a great need in the professional world on new, improved carriers for the introduction from boron in target tumor cells to boron neutron capture therapy and for imaging Purposes.

It is also known that certain hormones bind to and activate intracellular receptors so that the gene activity pattern in the cells changes. In the hormonal regulation of gene activity, a ligand (which can be a hormone or a synthetic analogue) penetrates a cell by facilitating diffusion. Once in the cell, the ligand binds to its intracellular receptor to form a ligand / intracellular receptor complex that induces a change in the shape of the receptor and activates the receptor so that it performs other functions in the cell. It is generally believed that the ligand / intracellular receptor complex has specific short DNA sequences within the Control region of the genes that respond to hormones is recognized and bound to it, thus mediating gene activity. Although much remains to be learned about the details of these mechanisms, it is known that exogenous triggers such as e.g. B. Hormones modulate gene transcription through concerted action with intracellular components, including intracellular receptors and discrete DNA, known to be hormone responsive elements. In particular, it is known that hormones such as the glucocorticoid, sex and thyroid hormones penetrate cells by facilitating diffusion. It is also known that the hormones then bind to specific receptor proteins and thus produce a hormone / receptor complex. It is believed that the binding of the hormone to the receptor causes an allosteric change in the receptor protein. As a result of this change, it is assumed that the hormone / receptor complex can bind with high affinity to certain specific sites on the chromatin DNA. These sites, which are given a variety of names in the art, modulate the expression (transcription of RNA) of nearby target gene promoters. In contrast to protein or peptide therapeutics, natural and synthetic ligands for intracellular receptors are small organic molecules that have many pleasant properties in common with pharmaceutical drugs, including their suitability for oral or topical administration. The receptors for the non-peptide hormones are closely related representatives of a protein superfamily. The receptors from this protein superfamily are called intracellular receptors because these receptors are located inside the target cells - in contrast to the receptors for neurotransmitters and protein or peptide hormones and growth factors that are located in the plasma membranes of cells.

Summary the invention

It has now been discovered that compounds containing boron can be introduced into hormonally responsive cells having at least one type of intracellular hormone receptor by contacting the cells with a compound as described in claim 1 or claim 7. The present invention provides a compound as described in the preamble of claim 1, wherein the ¹⁰ B units comprise at least eighteen ¹⁰ B atoms. Also provided is a compound as described in claim 7. The present invention also provides a method for introducing boron into cells containing intracellular hormone receptors as described in claim 10. The unit containing ¹⁰ B attaches to the hormone receptor of the cells, which can then be irradiated with neutrons in order to kill the cells by boron neutron capture. To avoid activating the cell outside the target areas of the radiation, the ligands according to the invention are antagonists which bind to the intracellular receptors but do not activate these intracellular receptors and which block activation of the receptors by subsequent action of the natural hormones of the receptors. In alternative embodiments, the target cells can be brought together with a boron-containing conjugate of the invention to attach boron to the target cells, and then the target cells can be imaged using, for example, magnetic resonance imaging techniques.

In typical embodiments of this aspect of the invention, the target cells may include, for example, metastatic breast cancer cells, and the boron neutron capture gene may comprise a ¹⁰ B moiety attached to a ligand such as e.g. B. is conjugated to a tamoxifen residue or an analog with binding specificity for estrogen receptors. For example, in other typical embodiments, the target cells may include prostate cancer cells, and the boron neutron capture agent may include a ¹⁰ B moiety attached to a ligand such as a 4'-substituted or 3 ', 4'-substituted anilide residue with binding specificity for androgen. Receptors is conjugated. For example, in further typical embodiments, the target may comprise leukemia cells, and the boron neutron capture gene may comprise a ¹⁰ B moiety conjugated to a ligand with binding specificity for glucocorticoid receptors; the target may comprise renal carcinoma cells and the boron neutron capture agent may comprise a unit containing ¹⁰ B conjugated to a ligand with binding specificity for progesterone receptors, androgen receptors or glucocorticoid receptors; the target cells may comprise endometrial cancer cells and the boron neutron capture agent may comprise a ¹⁰ B moiety conjugated to a ligand with binding specificity for estrogen receptors or progesterone receptors; the target cells may include ovarian carcinoma cells and the boron neutron capture gene may comprise a ¹⁰ B moiety conjugated to a ligand with binding specificity for estrogen receptors or progesterone receptors; or the target cells may include carcinoma cells of the cervix, vagina or vulva, and the boron neutron capture agent may comprise a ¹⁰ B moiety conjugated to a ligand with binding specificity for estrogen receptors or progesterone receptors.

In further aspects of the invention, novel compounds are provided which comprise a ¹⁰ B unit conjugated to a ligand with binding specificity for an intracellular receptor of a target cell. Typical embodiments of the compounds according to the invention include conjugates with Li ganden, the binding specificity for glucocorticoid and mineralocorticoid receptors; Sex steroid receptors such as progesterone, estrogen, estrogen-related and androgen receptors; as well as vitamin D3, thyroid and retinoic acid receptors. The conjugate unit containing ¹⁰ B contains at least eighteen ¹⁰ B atoms. Suitable boron neutron capture agents can be based on polyhedral borane anion derivatives, on derivatives which comprise two polyhedral borane anion cages which are linked to one another, to form a structure which comprises 20 boron atoms, on polyhedral carboranes, for example compounds of the formula closo- C ₂ B _n-2 H _n or closo-CB _n-1 H _n , on oligomeric peptides composed of boron-rich α-amino acids, or on boron-rich oligophosphates. Exemplary compounds of the invention or pharmaceutically acceptable salts thereof can be represented by the formula
Bo - [- (L) _m -Lig] _n
generally represent where Bo is a boron containing moiety comprising at least eighteen ¹⁰ B atoms, L is a linker group, Lig is a ligand, m is 0 or 1, and n is an integer greater than or equal to 1.

Short description of the drawings

The above aspects and others Advantages of this invention can be better understood from the following detailed Description and accompanying drawings, in which:

1A is a schematic representation of the synthetic route for the synthesis of the B ₁₀ flutamine derivative N- (closo-2-B ₁₀ H ₉ ) -N '- (3'-trifluoromethyl-4'-nitroaniline) urea 8 as described in Example 1;

1 B is a schematic representation of the synthetic route for the synthesis of the B ₂₀ flutamine derivative tetrasodium- (apical, apical-1- [2'-B ₁₀ H ₉ ] -2NH- [3 "-trifluoromethyl-4" -nitrophenyl] -B ₁₀ H ₈ 10 as described in Example 2;

2A is a schematic representation of the synthetic route for the synthesis of the closo-carboranyl oligophosphate-trailer-flutamine derivative 1-N- (closo-CB ₁₀ -hexylamine) -6-N- (3'-trifluoromethyl-4'-nitroaniline) suberyldiamid 13 as in Example 3 is described;

2 B is a schematic representation of the synthetic route for the synthesis of the nido-carboranyl oligophosphate-trailer-flutamine derivative 1-N- (nido-CB ₁₀ -hexylamine) -6-N- (3'-trifluoromethyl-4'-nitroaniline) subylenediamide 16 as in Example 4 is described;

3 a schematic representation of the synthetic route for the synthesis of the B ₂₀ tamoxifen derivative Na ₄ (apical, apical-1- [2'-B ₁₀ H ₉ ] -2- [N-cis-1- (4'-β-aminoethoxyphenyl) -1,2-diphenylbutene] -B ₁₀ H ₈ ) 22 as described in Example 5;

4 a schematic representation of the synthetic route for the synthesis of the closo-carboranyl oligophosphate-trailer tamoxifen derivative N ₁ - (closo-CB ₁₀ - hexylamine) -N ₆ - (cis-1- [4'-ß-aminoethoxyphenyl] -1.2 -diphenylbutene) sublenediamide 23 as described in Example 6;

5 is a schematic representation of the synthetic route for the synthesis of the nido-carboranyl oligophosphate-trailer tamoxifen derivative N ₁ - (nido-CB ₁₀ -hexylamine) -N ₆ - (cis-1- [4'-ß-aminoethoxyphenyl] -1.2 -Diphenylbuten) subenldiamid 24 as described in Example 7.

Full Description of the preferred embodiment

In accordance with the present invention, boron is introduced into intracellular hormone receptor-containing cells by bringing the cells together with a conjugate comprising a boron-containing moiety conjugated to a ligand with binding specificity for at least one type of intracellular hormone receptor of the cells, the boron-containing one Unit comprises at least eighteen ¹⁰ B atoms.

As with the present invention used is said to be "intracellular Cells Containing Receptors "for Cells stand that have one or more intracellular receptors for natural or contain synthetic hormones or hormone analogues. Especially with the present preferred embodiments can the receptors selected be from the super family of steroid hormone receptors, including the adrenal steroid receptors, such as glucocorticoid and mineralocorticoid receptors; Sex steroid receptors such as progesterone, estrogen, estrogen-related and androgen receptors; as well as vitamin D3, thyroid and retinoic acid receptors. See for example Evans et al., Science 240, 889ff (1988).

The boron-containing agents of the invention comprise a boron-containing moiety that comprises at least eighteen ¹⁰ B atoms and is conjugated to a ligand with binding specificity for an intracellular hormone receptor of the cells. The agents according to the invention can therefore be represented by the formula
Bo - [- (L) _m -Lig] _n
generally represent where Bo is a boron containing moiety comprising at least eighteen ¹⁰ B atoms, L is a linker group, Lig is an antagonist ligand, m is 0 or 1, and n is an integer greater than or equal to 1.

As used herein referred to the term boron-containing unit means the rest of a boron compound after reaction with a ligand to form a conjugate according to the invention.

darunter Verbindungen der Formel M_nH₂₀H₁₇L, worin M ein Kation ist, z. B. ein Alkalimetall- oder Tetraalkylammonium-Kation, n eine ganze Zahl von 1 bis 4 ist, und L ein Zweielektronendonor ist. Vorzugsweise ist M Na, K, Cs oder Rb, und Alkyl umfaßt Methyl, Ethyl und andere Alkyle, die das resultierende Salz nicht unlöslich machen. L ist ein Zweielektronendonor, vorzugsweise ausgewählt aus der Gruppe bestehend aus -NHR₁R₂, wobei R₁ und R₂ gleich oder verschieden sind und ausgewählt sind aus der Gruppe bestehend aus Wasserstoff, Benzyl, Alkyl und Diamin (z. B. Ethylendiamin); und -SR₁R₂, wobei R, ausgewählt ist aus der Gruppe bestehend aus Wasserstoff, Benzyl, Alkyl und Diamin, und R₂ ausgewählt ist aus der Gruppe bestehend aus Wasserstoff, Alkyl, -XCN, -XCO, -XNCO, -XCO₂OH, -XCO₂OR, -XNHCONBR₁, -XOOOH und -XCONHR₁, worin X Alkyl oder Arylalkyl mit 1 bis 20 Kohlenstoffatomen ist. Vorzugsweise ist L ausgewählt aus der Gruppe bestehend aus -NH₃, -NH₂-CH₂-Ph, -NH₂CH₂CH₂NH₂ und -NH₂(CH2)₇CH₃. Besonders bevorzugt ist L -NH₃.The boron compounds of the invention may comprise two polyhedral borane anion cages linked together to form a linked cage structure comprising, for example, 20 boron atoms (hereinafter referred to as "B ₂₀ compounds"; see MF Hawthorne, Angewandte Chemie) , Int. Edn. Engl. 32, 950-984 (1993)). The B ₂₀ compounds and derivatives thereof according to the invention can be represented by the general formulas:

including compounds of the formula M _n H ₂₀ H ₁₇ L, where M is a cation, e.g. An alkali metal or tetraalkylammonium cation, n is an integer from 1 to 4, and L is a two-electron donor. Preferably M is Na, K, Cs or Rb and alkyl includes methyl, ethyl and other alkyls which do not render the resulting salt insoluble. L is a two-electron donor, preferably selected from the group consisting of -NHR ₁ R ₂ , where R ₁ and R _{2 are the} same or different and are selected from the group consisting of hydrogen, benzyl, alkyl and diamine (e.g. ethylenediamine) ; and -SR ₁ R ₂ , wherein R is selected from the group consisting of hydrogen, benzyl, alkyl and diamine, and R _{2 is} selected from the group consisting of hydrogen, alkyl, -XCN, -XCO, -XNCO, -XCO ₂ OH, -XCO ₂ OR, -XNHCONBR ₁ , -XOOOH and -XCONHR ₁ , wherein X is alkyl or arylalkyl having 1 to 20 carbon atoms. L is preferably selected from the group consisting of -NH ₃ , -NH ₂ -CH ₂ -Ph, -NH ₂ CH ₂ CH ₂ NH ₂ and -NH ₂ (CH2) ₇ CH ₃ . L -NH ₃ is particularly preferred.

dargestellt, während sich die entsprechenden anionischen o-nido-C₂B₉H₁₂-Carborane sowie deren Derivate darstellen lassen als:

worin M^® ein Kation wie etwa ein Alkalimetallkation oder Tetraalkylammonium-Kation bedeutet.The boron compounds can also be based on polyhedral carboranes, for example compounds of the formula closo-C ₂ B _{n- 2} H _n (Dunks, G. et al., Accts. Chem. Res. 6, 124ff (1973); Grafstein, J Inorg. Chem. 2, 1128ff (1963)), or closo-CB _{n - 1} H _n ^- (Knoth, W., J. Am. Chem. Soc. 89, 1274ff (1967); Morris, J. et al ., Proceedings of the Fourth International Symposium on Neutron Capture Therapy of Cancer in Progress in Neutron Capture Therapy of Cancer; B. Allen et al., Ed., Plenum Press, New York, pp. 215ff (1992)), and on their corresponding nido derivatives, e.g. B. Compounds of the formula nido-C ₂ B _{n - 3} H _n ^- . The o-closo-C ₂ B ₁₀ H ₁₂ carboranes, including their derivatives, are generally defined by the structures

shown, while the corresponding anionic o-nido-C ₂ B ₉ H ₁₂ -carboranes and their derivatives can be represented as:

wherein M ^{® is} a cation such as an alkali metal cation or tetraalkylammonium cation.

worin M⁺ ein Alkalimetallkation oder Tetraalkylammonium wie vorstehend definiert ist, R₁ und R₂ unabhängig voneinander ausgewählt sind aus Gruppen, die zur Bindung an einen Liganden geeignet sind, zielgerichteten Einheiten und anderen funktionellen Gruppen, um den Verbindungen die gewünschten Eigenschaften zu verleihen, m eine ganze Zahl von 1 bis 10 ist, und n eine ganze Zahl von 2 bis 150, vorzugsweise 20 bis 100 ist. Wie vorstehend verwendet, soll die Gruppe

eine Carboranyl-Käfigstruktur mit folgender struktureller Konfiguration darstellen:

In a particularly preferred manner, the boron compounds according to the invention can comprise a plurality of carborane cage structures, for example oligomeric peptides, constructed from boron-rich α-amino acids with the aid of the Merrifield solid-phase synthesis methods as disclosed in Varadarajan, M. et al., Bioconj. Chem. 2, 242 (1991); and Paxton, R. et al., Bioconj. Chem. 3, 243 (1992), the disclosures of which are hereby mentioned by quotation. The boron-containing agents according to the invention can very particularly preferably comprise carborane-derived oligophosphates, for example those described in Kane, R. et al., "Automated Syntheses of Carborane-Derived Homogeneous Oligophosphates: Reagents for Use in the Immunoprotein-Mediated Boron Neutron Capture Therapy of Cancer ", J. Am. Chem. Soc. 115 (19), 8853-8854 (1993); Kane, R. et al., "Solution-Phase Synthesis of Boron-Rich Phosphates", J. Org. Chem. 58 (12), 3227-3228 (1993); and R. Kane et al., "Novel Carboranyl Diols and Their Derived Phosphate Esters", Advances in Neutron Capture Therapy, AH Soloway et al., ed., Plenum Press, New York (1993), the disclosures of which are hereby incorporated by reference , The typical compounds of this class include the compounds of the formula:

wherein M ^{+ is} an alkali metal cation or tetraalkylammonium as defined above, R ₁ and R _{2 are} independently selected from groups suitable for binding to a ligand, targeted units and other functional groups to impart the desired properties to the compounds, m is an integer from 1 to 10, and n is an integer from 2 to 150, preferably 20 to 100. As used above, the group

represent a carboranyl cage structure with the following structural configuration:

The isotopic content of the boron employed in the agents of the present invention can range from the natural abundance of approximately 19.78% ¹⁰ B up to an enriched fraction of 95% or more are of ¹⁰ B. When used for boron neutron capture therapy, a material that is highly enriched in ¹⁰ B is preferred.

Among those containing for conjugation to boron Compounds suitable ligands include non-peptide small ones organic molecular compounds, which are specific to the intracellular target receptors according to the invention can bind. Naturally occurring hormones or agonists of naturally occurring hormones bind to the intracellular Target receptors and solve Change of form and function in intracellular Receptors. Pure agonists such as estrogen and Progesterone specifically targets its corresponding receptors, however the use of pure agonists in the practice of the invention Stimulate the growth of certain tumor cells, whether now inside or outside the tissue area that should be the target of neutron radiation. Because tumor cells outside the target area of neutron radiation by no cell killing Boron neutron capture, the use of pure agonists during execution of the invention in some cases to stimulate tumor growth in more distant places to lead. Accordingly, they are ligands according to the invention Antireceptor agents or antagonists that target the intracellular receptors bind, however, this intracellular Do not activate receptors, and activate the receptors through subsequent exposure to the natural hormones of the receptors To block. Antagonists for intracellular Receptors are known to the person skilled in the art.

worin Bo eine ¹⁰Bor enthaltende Einheit ist, L eine Linker-Gruppe ist, und n 0 oder 1 ist, oder die pharmazeutisch annehmbaren Salze derselben.For example, if the intracellular target receptors are androgen receptors, the typical ligands of the invention include antiandrogenic agents, such as the antiandrogenic N-phenylarnide derivatives disclosed in US Pat. Nos. 3,995,060, 4,191,775, 4,329,364, 4,386 080, 4 636 505, 4 474 813 and 4 921 941, and in EP publication No. 0 253 503 B1, the disclosures of which are hereby incorporated by reference Quote should be mentioned. A currently particularly preferred antiandrogenic agent for this purpose is 2-methyl-N- [4-nitro-3- (trifluoromethyl) phenyl] propanamide, also known as flutamide. If, as a further illustrative example of an embodiment of the invention, the ligand is a flutamide residue, the boron-containing compounds according to the invention with binding specificity for androgen receptors include the compounds of the formula:

wherein Bo is a unit containing ¹⁰ boron, L is a linker group, and n is 0 or 1, or the pharmaceutically acceptable salts thereof.

Are - as further explanatory Example - the intracellular Target receptors estrogen receptors are among the typical ligands according to the invention Antiestrogen agents, such as the antiestrogenic triphenylalkene derivatives, which are disclosed in U.S. Patent Nos. 4,198,435, 4,206,234, 4 307 111, 4 310 523, 4 623 660, 4 839 155, 5 047 431, 5 192 525 and 5 219 548, the disclosures of which are hereby mentioned by quotation. To the present particularly preferred antiestrogenic ligands of this type include Example 1- (p-β-dimethylaminoethoxyphenyl) trans-1,2-diphenyl-2-pheny-but-1-ene, also known as tamoxifen; 1- [2- [p- (3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl) phenoxy] ethyl] pyrrolidine, also known as nafoxidin; and 2- [p- (2-chloro-1,2-diphenylvinyl) phenoxy] triethylamine, also known as clomiphene, or the pharmaceutically acceptable Salts thereof.

In addition to those described above Androgen and estrogen antagonists are suitable ligands also antagonists for Adrenal steroid receptors, such as glucocorticoid and mineralocorticoid receptors, vitamin D3 receptors, thyroid receptors and retinoic acid receptors, such as those skilled in the art will be clear.

The boron-containing agents of present invention can either as free agents or in conjunction with other delivery vehicles such as such as single-layer or multi-layer vesicles, including Liposomes. Production and use of vesicles in drug delivery applications are known in the art and documented. See e.g. B. Shelly, K. et al., PNAS 89, 9039ff (1992), the disclosure of which is hereby mentioned by quotation. In one embodiment according to the invention, from Liposomes encapsulated tumor treatment agents according to the invention comprise single-layer or multi-layer liposome, wherein the Liposome at least one encapsulating double layer and one through the encapsulating double layer includes defined interior. On Agent containing boron according to the invention can then be in this interior or between the layers of the liposome be encapsulated. An abundance of lipid particles Form delivery vesicles useful in this aspect of the invention. For example, you can Phospholipid vesicles, such as those in EP patent application 0 272 091 disclosed, the disclosure of which is not mentioned here by quotation, used with the boron-containing agents of the present invention become. These phospholipid vesicles are composed of one single encapsulating phospholipid membrane attached to an amphiphilic associated substrate is attached. Let liposomes of this type are manufactured using methods that are common in the professional world. For example, a hydrated phospholipid suspension forms in the stir up multilayer vesicles, with many double layers with diameters of 1-10 μm through water are separated. By using a shear or homogenizing Force, such as sonication, on a multi-layer suspension produces small single-layer vesicles of a size that range from about 30 to about 250 nm, and preferably from about 50 to about 100 nm average diameter. This range is preferred deemed to have optimal in vivo cycle times and specificity for the target cell to obtain. As used herein, "monolayer" is intended in general one to three double layers and preferably one double layer rewrite. The average diameter of the liposome encapsulated according to the invention Agents containing borane depend depends on many factors, including the sonication time of the phospholipid suspension, the composition of the lipid substances used, the processes, with the help of which the liposome is produced, and other relevant Factors. Liposomes, the boron-containing agents according to the invention embedded in the double layer of the liposomes can be obtained using the methods disclosed by M.F. Hawthorne, Applied Chemistry, Int. Edn. Engl. 32, 950-984 (1993). For example a dried film can be made by dissolving a lipophilic boron according to the invention containing agent and a cholesterol / phospholipid mixture in Chloroform and subsequent Remove the solvent in a vacuum. The resulting dry film is then sonicated homogenized, and the vesicles formed are free of boron Agent separated, for example by elution through a Sephadex G 25 column (medium) with isotonic phosphate-buffered saline or lactose. The in the The amount of boron embedded in the liposome double layer depends on that of the original Mix added amount of boron-containing agent and can control as needed.

The agents containing boron according to the invention can to kill in vitro or in vivo or imaging target cells are used, the intracellular receptors contain. To kill target cells through boron neutron capture become the cells with at least one boron-containing Agents are brought together, and then the cells become one Neutron bombardment exposed from a source that is thermal, epithermal or fast neutrons are emitted, as is known to the person skilled in the art. For in vivo applications include the compositions of the boron-containing agents according to the invention in generally a lot of boron when administered to a human or an animal is effective in that a sufficient amount of boron is delimited at the points of the target tissue in order to subsequently neutron radiation, Boron neutron capture and killing of target cells or a visual representation of the target tissue enable, along with a pharmaceutically acceptable carrier. For this purpose, in general any pharmaceutically acceptable carrier can be used as long as this carrier the stability or bioavailability the boron-containing invention Agents not affected.

For boron imaging of target cells the cells become more similar Brought together with at least one boron-containing agent according to the invention, and the cells or tissues are imaged using imaging Magnetic resonance techniques, such as those described are described in Kabalka, G.W. et al., "A New Boron MRI Method For Imaging BNCT Agents in Vivo ", in Progress in Neutron Capture Therapy For Cancer, B.J. Allen et al., Ed., Plenum Press, New York, NY 1992, Pp. 321-323; and Bradshaw, K.H. et al., "In Vivo Pharmacokinetic Evaluation of Boron Compounds Using Magnetic Resonance Compounds in Spectorscopy and Imaging "in Progress In Neutron Capture Therapy For Cancer, B. J. Allen et al., Ed., Plenum Press, New York, NY 1992, pp. 325-329.

The compositions with the boron containing agent can in any effective, pharmaceutically acceptable form to warm-blooded animals, including humans, as well as to other animals z. B. in topical, rinsing, oral, suppository, parenteral or infusible dosage forms as a topical, buccal, or nasal spray or in any other way administered, which is effective in that the boron containing agents are supplied to a site with target cells. The route of administration is preferably such that delivery and delimitation of the agents on or to the target cells become.

For Topical applications can be the pharmaceutically acceptable carrier Form of liquids, creams, Accept lotions or gels and also organic solvents, Emulsifiers, gelling agents, moisturizing agents, stabilizers, Surfactants, wetting agents, preservatives, long-term release agents and smaller amounts of humectants, complexing agents, dyes, perfumes and other ingredients commonly found in pharmaceuticals Compositions for topical administration can be used.

Compositions for injection can be determined pharmaceutically acceptable sterile aqueous or non-aqueous solutions, Suspensions or emulsions include. To the examples of suitable non-aqueous vehicles, diluents, solvent or count vehicle Propylene glycol, polyethylene glycol, vegetable oils such as olive oil as well injectable organic esters such as B. Ethyl oleate. These compositions can also auxiliaries such as preservatives, wetting agents, Emulsifiers and dispersants include. They can be sterilized for example by filtration through a bacteria-retentive Filters or by incorporating sterilizing agents into the compositions. You can can also be produced in the form of sterile solid compositions, the prior to administration in sterile water, sterile saline or dissolved or suspended in other sterile injectable media can.

To the fixed dosage forms for oral or topical administration Capsules, tablets, pills, suppositories, powders and granules. With fixed dosage forms the compositions with at least one inert diluent such as sucrose, lactose or starch can be mixed and additional lubricants, Buffering agents, enteric coatings and other ingredients include those known to those skilled in the art.

The actual dosage levels of the Agents containing boron in the compositions according to the invention can thus changed that quantities on boron-containing agent at the location of the target cells, in particular tumor cells, are obtained which are effective in that the desired therapeutic, prophylactic or diagnostic response is achieved. Accordingly judges the chosen one dosage level according to the nature and location of the target cells, the desired ones Amount of boron required for effective neutron capture or for imaging Purpose of the target cells is required, the nature and the boron content of the boron-containing agents used, the route of administration and other factors. Usually a sufficient amount of the boron-containing agent used to a boron content of at least about 1 µg / g Target cell tissue, more preferably at least about 10 µg / g target cell tissue and particularly preferably to achieve at least about 20 μg / g of target cell tissue.

The above can be said better understand in conjunction with the following representative Examples that are used for illustration and are not to be taken as limiting should.

EXAMPLES

example 1

Synthesis of a B ₁₀ flutamine derivative

N- (closo-2-B ₁₀ H ₉ ) -N '- (3'-trifluoimethyl-4'-nitroaniline) urea 8

The following synthesis can best be seen from 1A understand. Sodium hydride (60% suspension in mineral oil, 0.32 g, 8 mmol) is placed in an oven-dried 25 ml round bottom flask with a magnetic stirrer, septum and nitrogen inlet. The gray powder is washed with 5 x 10 ml of dry hexane to remove the mineral oil by syringe, then 10 ml of dry tetrahydrofuran is added and the mixture is cooled to 0 ° C with an ice / water bath. A solution of 5-amino-2-nitrobenzotrifluoride (2, 1.53 g, 7.5 mmol) in 5 ml of dry tetrahydrofuran is added dropwise with a syringe. The ice / water bath is then removed and the resulting solution is stirred at room temperature for four hours, at which point the evolution of hydrogen has ceased. The mixture is again placed in the EisNVasser bath and a solution of compound 7 (closo-2-B ₁₀ H ₉ NCΟ ² · [Et ₃ NH ⁺ ] ₂ ; Shelly, K. et al., Inorg. Chem 31 (13), 2899-2892 (1992); 0.91 g, 2.5 mmol) in 5 ml of dry tetrahydrofuran added dropwise over 10 min. The ice / water bath is then removed and the mixture is stirred at room temperature for 24 hours.

The volatile components are then removed under vacuum, and the residue is dissolved in 2 ml dichloromethane / ethanol (8: 2 VoL / oI.) Solved. This solution is about a column chromatographed (60A, 230-400 mesh Silica gel, column 20 cm × 2 cm), whereby dichloromethane / ethanol (gradient 8: 2 to 1: 1 VoLNoI. over 500 ml) is used and 10 ml fractions are collected. First of all the excess amine 2 eluted, followed by the desired one Product B. The 8 containing fractions are combined and the solvent stripped under vacuum. Recrystallization from acetone / pentane (1: 1 vol / vol.) Results in the pure compound B.

Example II

Synthesis of a B ₂₀ flutamine derivative

Tetrasodium- (apical, apical-1- [2'-B ₁₀ H ₉ ] -2-N H- [3 "-trifluoromethyl-4" -nitrophenyl] -B ₁₀ Η ₈ 10

The following synthesis can best be seen from 1 B understand. Sodium hydride (0.10 g, 60% suspension in mineral oil, 2.5 mmol) is placed in an oven-dried 25 ml round bottom flask with a magnetic stirrer, septum and nitrogen inlet. The gray powder is washed with 5 x 10 ml of dry hexane to remove the mineral oil by syringe, then 10 ml of dry tetrahydrofuran is added and the mixture is cooled to 0 ° C with an ice / water bath. A solution of 5-amino-2-nitrobenzotrifluoride (2, 0.51 g, 2.5 mmol) in 5 ml of dry tetrahydrofuran is added dropwise with a syringe. Then the ice / water bath is removed, the resulting solution is stirred for four hours at room temperature, and at this point the evolution of hydrogen has stopped. The mixture is again placed in the ice / water bath and a solution of compound 9 (n - B ₁₀ H ₁₈ ^2- · [Et ₄ N ⁺ ] ₂ ; Hawthorne, MF et al., J. Am. Chem. Soc. 85, 3704 (1963); Hawthorne, MF et al., J. Am. Chem. Soc. 87, 4740-4746 (1965); 1.24 g, 2.5 mmol) in 5 ml of dry tetrahydrofuran at once added. The ice / water bath is then removed and the mixture is stirred at room temperature for 24 hours (Feakes, DA et al., Proc. Natl. Acad. Sci., USA, 91, 3029-3033 (1994)).

The volatile components are then stripped off in vacuo and the residue becomes anhydrous in 5 ml Ethanol dissolved. The product is made by felling with a saturated solution isolated from tetraethylammonium bromide in anhydrous ethanol, to the raw product in the form of a mixture of api cal, equatorial and to give apical, apical bound isomers. This solid will in 10 ml of anhydrous acetonitrile in a dry 25 ml round bottom flask with magnetic stirrer and inlet for dry Nitrogen dissolved, and 0.5 ml of freshly distilled trifluoroacetic acid are added. The resulting solution is stirred for 2 hours at room temperature, and then acetonitrile and trifluoroacetic acid stripped under vacuum. By recrystallizing the residue (from acetone / pentane, 8: 2 vol / vol.) the pure compound is obtained 10 as tetrakis (tetraethylammonium) salt. This salt is in one 1: 1 mixture of acetonitrile / water (v / v) dissolved and on an ion exchange column given in the sodium form (2 × 20 cm, Dowex 50W-A8, pre-equilibrated with 200 ml of a 25:75 mixture (vol / vol.) of acetonitrile / water). The sample is then mixed with 200 ml of a 25:75 mixture (VoLNoI.) from acetonitrile / water from the column eluted, collecting 10 ml fractions. The product-containing ones Fractions are pooled and dried under vacuum to remove the Tetrasodium salt 10 to give.

Example III

Synthesis of a closo-carboranyl oligophosphate-trailer-flutamine derivative

1-N- (closo-CB ₁₀ -hexylamine) -6-N- (3'-trifluoromethyl-4'-nitroaniline) suberyldiamide 13

The following synthesis can best be seen from 2A understand.

The closo-CB ₁₀ -hexylamine (11; Kane, RR et al., J. Am. Chem. Soc. 155, 8853-8854 (1993); 70 mg, 20 μmol) is in a 10 ml centrifuge tube in 50 mM Sodium borate buffer, pH 7.0, dissolved to a final concentration of 10 mM (2 ml). A 200 mM solution of disuccinimidyl suberate (DSS; Pierce, Inc.) in ice-cold dimethylformamide (DMF, 1 ml, 200 μmol) is prepared in a 1.5 ml microcentrifuge tube and added to the closo-CB ₁₀ -hexylamine. The centrifuge tube is then at room temperature 2 Slightly spun for hours. A 7 A ml portion of ethyl acetate (pre-saturated with 50 mM sodium borate buffer, pH 7.0) is added to the centrifuge tube and the two-phase mixture is swirled vigorously for one minute to ensure complete mixing. After standing undisturbed for 3 minutes at room temperature to allow the layers to separate completely, the ethyl acetate containing the unreacted DSS is decanted from the centrifuge tube. This process is repeated four more times to ensure complete separation of the crosslinker (the desired activated ester, compound 12, is retained in the aqueous layer).

Compound 2 (5-amino-2-nitrobenzotrifluoride, 20 mg, 100 μmol) is dissolved in 100 μl ice-cold DMF and added to the aqueous solution of 12 in the centrifuge tube. The resulting milky suspension is swirled at room temperature for 6 hours. The entire reaction mixture is then placed on a PD-10 column (Pharmacia, 10 ml gel bed of Sephadex G-25, pre-equilibrated with 50 ml Milli-Q water) and the product, conjugate 13 , is eluted with 20 ml of distilled water, collecting 0.5 ml fractions. The solvent is removed from the fractions containing the product to give 13 as a white powder.

Example IV

Synthesis of a nido-carboranyl oligophosphate-trailer-flutamine derivative

1-N- (nido-CB ₁₀ -hexylamine) -6-N- (3'-trifluoromethyl-4'-nitroaniline) subylenediamide 16

The following synthesis can best be seen from 2 B understand. The nido-CB ₁₀ -hexylamine (14; Kane, RR et al., J. Am. Chem. Soc. 155, 8853-8854 (1993); 70 mg, 20 μmol) is in a 10 ml centrifuge tube in 50 mM Sodium borate buffer, pH 7.0, dissolved to a final concentration of 10 mM (2 ml). A 200 mM solution of disuccinimidyl suberate (DSS; Pierce, Inc.) in ice-cold dimethylformamide (DMF, 1 ml, 200 μmol) is prepared in a 1.5 ml microcentrifuge tube and added to the nido-CB ₁₀ -hexylamine. The centrifuge tube is then gently spun at room temperature for 2 hours. A 7 ml portion of ethyl acetate (pre-saturated with 50 mM sodium borate buffer, pH 7.0) is added to the centrifuge tube and the two phase mixture is vortexed vigorously for one minute to ensure thorough mixing. After standing undisturbed at room temperature for 3 minutes to allow the layers to separate completely, the ethyl acetate containing the unreacted crosslinking is decanted from the centrifuge tube. This process is repeated four more times to ensure complete separation of the DSS (the desired activated ester, compound 15, is retained in the aqueous layer).

Compound 2 (5-amino-2-nitrobenzotrifluoride, 20 mg, 100 μmol), is in 100 ul ice cold DMF dissolved and the aqueous solution of 15 in the centrifuge tube added. The resulting milky suspension is 6 hours long twisted at room temperature. The entire reaction mixture is then placed on a PD-10 column (Pharmacia, 10 ml gel bed of Sephadex G-25, pre-equilibrated with 50 ml Milli-Q water) and the product is eluted with 20 ml of distilled water, collecting 0.5 ml fractions. The solvent is subtracted from the fractions containing product to make 16 as white To give powder.

Example V

Synthesis of a B ₂₀ tamoxifene derivative

Na ₄ (apical, apical-1- [2'-B ₁₀ H ₉ ] -2- [N-cis-1- (4'-β-aminoethoxyphenyl) -1,2-diphenylbutene] -B ₁₀ H ₈ ) 22

The following synthesis can best be seen from 3 understand. Sodium hydride (0.10 g, 60% suspension in mineral oil, 2.5 mmol) is placed in an oven-dried 25 ml round bottom flask with a magnetic stirrer, septum and nitrogen inlet. The gray powder is washed with 5 × 10 ml of dry Nexan by syringe to remove the mineral oil, then 10 ml of dry tetrahydrofuran are added and the mixture is cooled to 0 ° C. with an ice / water bath. A solution of cis-1- (4'-β-aminoethoxyphenyl) -1,2-diphenylbutene (17, 0.89 g, 2.5 mmol) in 5 ml of dry tetrahydrofuran is added dropwise with a syringe. Then the ice / water bath is removed, the resulting solution is stirred for four hours at room temperature, and at this point the evolution of hydrogen has stopped. The mixture is again placed in the ice / water bath and a solution of compound 9 (n-B ₂₀ Η ₁₈ 2- · [Et ₄ N ⁺ ] ₂ ; Nawthorne, M. F: et al., J Am. Chem. Soc. 85, 3704 (1963); Hawthorne, MF et al., J. Am. Chem. Soc. 87, 4740-4746 (1965); 1.24 g, 2.5 mmol) in 5 ml of dry tetrahydrofuran added at once. The ice / water bath is then removed and the mixture is stirred at room temperature for 24 hours (Feakes, DA et al., Proc. Natl. Acad. Sci., USA, 91, 3029-3033 (1994)).

The volatile components are then stripped off in vacuo and the residue becomes anhydrous in 5 ml Ethanol dissolved. The product is cut through with a saturated solution isolated from tetraethylammonium bromide in anhydrous ethanol, to the raw product in the form of a mixture of apical, equatorial and to give apical, apical bound isomers. This solid will in 10 ml of anhydrous acetonitrile in a dry 25 ml round bottom flask with magnetic stirrer and inlet for dry Nitrogen dissolved, and 0.5 ml of freshly distilled trifluoroacetic acid are added. The resulting solution is stirred for 2 hours at room temperature, and then acetonitrile and trifluoroacetic acid stripped under vacuum. By recrystallizing the residue (from acetone / pentane, 8: 2 VoLNoI.) the pure compound is obtained 22 as the tetrakis (tetraethylammonium) salt. This salt is in one 1: 1 mixture of AcetonitriI / water (Vol / Vol.) Dissolved and on an ion exchange column in the Given sodium form (2 × 20 cm, Dowex 50W-A8, pre-equilibrated with 200 ml of a 25:75 mixture (VoI./Nol.) from acetonitrile / water). The sample is then mixed with 200 ml of a 25:75 mixture (v / v) from acetonitrile / water from the column eluted, collecting 10 ml fractions. The product-containing ones Fractions are pooled and dried under vacuum to remove the Tetrasodium salt 22 to give.

Example VI

Synthesis of a closo-carboranyl oligophosphate-trailer-tamoxifen derivative

N ₁ - (closo-CB ₁₀ -hexylamine) -N ₆ - (cis-1- [4'-β-aminoethoxyphenyl] -1,2-diphenylbutene) suberyldiamide 23

The following synthesis can best be seen from 4 understand.

The closo-CB ₁₀ -hexylamine (11; Kane, RR et al., J. Am. Chem. Soc. 155, 8853-8854 (1993); 70 mg, 20 μmol) is in a 10 ml centrifuge tube in 50 mM Sodium borate buffer, pH 7.0, dissolved to a final concentration of 10 mM (2 ml). A 200 mM solution of disuccinimidyl suberate (DSS; Pierce, Inc.) in ice-cold dimethylformamide (DMF, 1 ml, 200 μmol) is prepared in a 1.5 ml microcentrifuge tube and added to the closo-CB ₁₀ -hexylamine. The centrifuge tube is then at room temperature 2 Slightly spun for hours. A 7 ml portion of ethyl acetate (pre-saturated with 50 mM sodium borate buffer, pH 7.0) is added to the centrifuge tube and the two-phase mixture is vigorously vortexed for one minute to ensure thorough mixing. After standing undisturbed for 3 minutes at room temperature to allow the layers to separate completely, the ethyl acetate containing the unreacted DSS is decanted from the centrifuge tube. This process is repeated four more times to ensure complete separation of the crosslinker (the desired activated ester, compound 12, is retained in the aqueous layer).

Compound 17 (cis-1- (4'-β-aminoethoxyphenyl) -1,2-diphenylbutene, 36 mg, 100 μmol), is ice cold in 100 μ1 DMF solved and the aqueous solution of 12 in the centrifuge tube added. The resulting milky suspension is 6 hours long at room temperature. The entire reaction mixture is then on a PD-10 column (Pharmacia, 10 ml gel bed of Sephadex G-25, pre-equilibrated with 50 ml Milli-Q water) and the product, conjugate 23, is distilled with 20 ml Water elutes, collecting 0.5 ml fractions. The solvent is subtracted from the fractions containing product to 23 as white To give powder.

Example VII

Synthesis of a nido-carboranyl oligophosphate-trailer tamoxifen derivative

N ₁ - (nido-CB ₁₀ -hexylamine) -N ₆ - (cis-1- (4'-β-aminoethoxyphenyl] -1,2-diphenylbutene) suberyldiamide 24

The following synthesis can best be seen from 5 understand.

The nido-CB ₁₀ -hexylamine (14; Kane, RR et al., J. Am. Chem. Soc. 155, 8853-8854 (1993); 70 mg, 20 μmol) is in a 10 ml centrifuge tube in 50 mM Sodium borate buffer, pH 7.0, dissolved to a final concentration of 10 mM (2 ml). A 200 mM solution of disuccinimidyl suberate (DSS; Pierce, Inc.) in ice-cold dimethylformamide (DMF, 1 ml, 200 μmol) is prepared in a 1.5 ml microcentrifuge tube and added to the nido-CB ₁₀ -hexylamine. The centrifuge tube is then gently spun at room temperature for 2 hours. A 7 ml portion of ethyl acetate (pre-saturated with 50 mM sodium borate buffer, pH 7.0) is added to the centrifuge tube and the two-phase mixture is vortexed vigorously for one minute to ensure thorough mixing. After standing undisturbed for 3 minutes at room temperature in order to allow the layers to separate completely, the ethyl acetate containing the unreacted crosslinker is decanted from the centrifuge tube. This process is repeated four more times to ensure complete separation of the DSS (the desired activated ester, compound 15, is retained in the aqueous layer).

Compound 17 (cis-1- (4'-β-aminoethoxyphenyl) -1,2-diphenylbutene, 36 mg, 100 μmol), is ice cold in 100 μ1 DMF solved and the aqueous solution of 15 in the centrifuge tube added. The resulting milky suspension is 6 hours swirled for long at room temperature. The entire reaction mixture is then on a PD-10 column (Pharmacia, 10 ml gel bed of Sephadex G-25, pre-equilibrated with 50 ml Milli-Q water) and the product is eluted with 20 ml of distilled water, collecting 0.5 ml fractions. The solvent is withdrawn from the product-containing fractions to 24 as a white powder to surrender.

Claims (25)

A compound comprising a ¹⁰ B unit conjugated to an antagonist ligand with binding specificity for an intracellular hormone receptor, characterized in that the ¹⁰ B unit comprises at least eighteen ¹⁰ B atoms. The compound of claim 1, wherein the ¹⁰ B unit comprises at least two polyhedral carboranes or derivatives thereof. The compound of claim 1, wherein the ¹⁰ B moiety comprises at least two polyhedral borane anion cages that are linked together to form a linked cage structure. The compound of claim 1, wherein the ¹⁰ B unit comprises multiple carborane cage structures. The compound of claim 1, wherein the ¹⁰ B unit comprises carborane-derivatized oligophosphates. A compound according to any preceding claim, wherein the intracellular receptor selected is from the group consisting of estrogen receptors, progesterone receptors, Androgen receptors and glucocorticoid receptors. Connection of the formula:

wherein Bo is a ¹⁰ B unit comprising at least eighteen ¹⁰ B atoms, L is a linker group, and n is 0 or 1, or a pharmaceutically acceptable salt thereof. The compound of claim 7, wherein the ¹⁰ B-containing moiety is derived from carboranes and multimers or polymers thereof. A compound according to claim 7 or 8, wherein L is CH ₂ and n is 1. In vitro procedures for introduction of boron in cells that contain intracellular hormone receptors comprising bringing the cells together with a compound one of the claims 1 to 9. The method of claim 10, wherein the cells are tumor cells. The method of claim 10 or 11, wherein the cells are metastatic breast cancer cells and the compound comprises a ¹⁰ B moiety conjugated to a ligand with binding specificity for estrogen receptors. The method of claim 12, wherein the compound comprises a ¹⁰ B moiety conjugated to tamoxifen. The method of claim 10 or 11, wherein the cells are prostate cancer cells and the compound comprises a ¹⁰ B moiety conjugated to a ligand with binding specificity for androgen receptors. The method of claim 14, wherein the ligand comprises a ¹⁰ B moiety conjugated to a 4'-substituted or 3,4'-disubstituted anilide residue. The method of claim 15, wherein the anilide residue is the 4'-nitro-3'-trifluoromethylisobutyranilide residue is. The method of claim 14, wherein the compound comprises a ¹⁰ B moiety conjugated to a cyproterone acetate residue. The method of claim 10 or 11, wherein the cells are leukemia cells and the compound comprises a ¹⁰ B moiety conjugated to a ligand with binding specificity for glucocorticoid receptors. The method of claim 10 or 11, wherein the cells are renal carcinoma cells and the compound comprises a ¹⁰ B moiety conjugated to a ligand with binding specificity for progesterone receptors, androgen receptors or glucocorticoid receptors. The method of claim 10 or 11, wherein the cells are endometrial carcinoma cells and the compound comprises a ¹⁰ B moiety conjugated to a ligand with binding specificity for estrogen receptors or progesterone receptors. The method of claim 10 or 11, wherein the cells are ovary carcinoma cells and the compound comprises a ¹⁰ B moiety conjugated to a ligand with binding specificity for estrogen receptors or progesterone receptors. The method of claim 10 or 11, wherein the cells are cervical, vaginal or vulvar carcinoma cells and the compound comprises a ¹⁰ B moiety conjugated to a ligand with binding specificity for estrogen receptors or progesterone receptors. In vitro method for imaging target cells that have intracellular hormone receptors included, comprising bringing the cells together with a compound according to one of the claims 1 to 9 and the subsequent one Formation of an image of the magnetic resonance of the cells associated Bors. Connection according to the claims 1 to 9 for use in killing of cells that have intracellular hormone receptors included with the help of boron neutron capture therapy. Use of compounds according to claims 1 to 9 for the preparation a boron neutron capture gene to kill cells that have intracellular hormone receptors contain.